Research methodology in protein chemistry, molecular biology, immunology and physiology will be applied to a receptor system relevant to psychosis. This will complement Dr. Javitch's previous training in pharmacology, neuroscience, and clinical psychiatry, and thereby facilitate his transition to an independent investigator in the basic science of psychiatric disorders. In addition to his research training, Dr. Javitch will continue a small commitment to clinical practice and teaching. The training supported by the SDA will prepare him to apply a molecular approach to studying the role of receptors in psychiatric illness. Three types of D2-like receptor (D2, D3 and D4) have been identified by cloning and sequencing. The pharmacology of these receptor types is similar, except for several differences which may have profound clinical ramifications. It is unclear which type, or combinations of types, is relevant to the etiology and treatment of psychosis, and the clinically relevant pharmacology cannot be inferred from the sequences alone. The specific intent of this proposal is to develop an understanding of how the structures of the D2, D3 and D4 receptors that form the binding sites for agonists, typical neuroleptics and atypical neuroleptics will be identified. D2 receptor will be prepared from bovine striatum, and D2, D3 and D4 receptor will be expressed using the baculovirus expression system. Specific residues of the receptor types will be radiolabeled by covalent modification, and the protein will be cleaved. The fragments will be separated and identified with polyclonal antibodies produced to synthetic peptides unique to each fragment. Specific residues will be identified by protein sequencing. These results will provide a basis for further analysis of the identified sites by site-directed mutagenesis and expression in cell lines, thereby laying a foundation for understanding the important pharmacological and physiological differences among the types of the dopamine D2-like receptors.
| Javitch, J A; Fu, D; Chen, J (1996) Differentiating dopamine D2 ligands by their sensitivities to modification of the cysteine exposed in the binding-site crevice. Mol Pharmacol 49:692-8 |
| Javitch, J A; Fu, D; Chen, J (1995) Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. Biochemistry 34:16433-9 |
| Javitch, J A; Fu, D; Chen, J et al. (1995) Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method. Neuron 14:825-31 |
| Javitch, J A; Li, X; Kaback, J et al. (1994) A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice. Proc Natl Acad Sci U S A 91:10355-9 |
